Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm. The loss of Chl during the senescence of cereal leaves is preceded by a gradual decline in protein content. It has been suggested that proteases responsible for the degradation of the proteins are instrumental in regulating senescence progress (10, 13). Thus, in detached oat leaves senescing in the dark, Martin and Thimann (15) observed increases in the activity of the two major proteases, with pH optima at 4.5 ('acidic' protease) and 7.5 ('neutral' protease). However, depending on the conditions of incubation of detached leaves, protein breakdown can occur at the same rate whether protease activity increases or not. Although protease activity may increase during leaf senescence, young leaf cells appear to already possess sufficient protease activity to degrade all proteins present (26). Thus, we proposed that in those cells the proteases are spatially separated from their protein substrates. During senescence, compartmentalization might be lost, leading to the accelerated loss of protein. According to this hypothesis, spatial contact between substrates and proteases rather than protease levels or activity would be the mechanism regulating the rate of protein degradation and, hence, senescence. Protease activities have been detected in isolated vacuoles of many plant species (3), including wheat (14, 29, 30) and barley (11, 21). The presence of endopeptidases and carboxypeptidases inside the vacuole suggests that the vacuolar sap is involved in the digestion of cytoplasmic proteins (18, 27). The proteins to I Present address: Research Institute ITAL, P. 0. Box 48, 6700 AA Wageningen, The Netherlands. be degraded are located in the cytoplasm and in organelles such as chloroplasts and mitochondria and are thus spatially separated from the proteolytic enzymes. So far, it is unknown how protein degradation during leaf senescence is controlled and to what extent vacuolar proteases participate in this process. Cytoplasmic constituents might be engulfed by the vacuole, in a process resembling endocytosis, and degraded by the vacuolar enzymes (18). Degradation of endogenous proteins inside isolated vacuoles of Acer pseudoplatanus has been reported by Canut et al. (6). These authors suggested that the specificity of the degradation is due to a selective transfer of proteins from the cytoplasm into the vacuole rather than selective proteolysis inside the vacuole (7). However, the mechanism of transport of proteins into the vacuole is not understood. On the other hand, protein degradation may occur in the cytosol and in plant organelles after proteins have been marked by ubiquitin (28). Alternatively, proteases synthesized and compartmentalized during early leaf development might be gradually liberated into the cytoplasm, as initially suggested by Balz (1). Thus, senescence might be controlled by passage of proteases out of the vacuole. Under conditions where protease activity does not increase during senescence, the relative amount of protease present in vacuoles is then expected to decrease during leaf development. Up to now, there is no evidence that vacuolar proteases are released into the cytosol, except when, in a final stage of senescence, lysis of the whole vacuole occurs (17). Recently, we obtained representative vacuoles from oat leaves at various stages of development (24) and observed that the vacuolar membrane remained physically intact up to an advanced stage of senescence, when more than 50% of the leaf protein had already been degraded. These data offered the possibility to prove the subcellular localization of the two major proteases of oat leaves up to this stage and to address the question as to how far vacuolar proteases can be involved in cytoplasmic protein degradation. MATERIAL AND METHODS Plant Material. Oat plants (Avena sativa L. cv Victory) were grown in pot soil in a growth cabinet as described previously (24). For comparison, plants grown in a greenhouse (26) were also used. To establish the subcellular distribution of the proteases throughout leaf development, first leaves were harvested at regular intervals. The distal 4.5 cm of the leaves were homogenized in a mortar with pestle in extraction medium containing 50 mM Tris, 0.2% ascorbic acid (w/v), 0.1% mercaptoethanol (v/v), 2 mm EDTA, and 3.5% NaCl. The pH was set at 8.0 with HCl. The tissue/buffer ratio was 4 g/15 ml (final volume). Fractionation. Subcellular fractionation was carried out by successive centrifugations of the extract at 1,000, 15,000, and 30,000g. Each pellet was washed once by gentle resuspension in extraction medium, after which the centrifugation step was repeated. The supernatant thus obtained was combined with the previous one, 536 www.plantphysiol.org on January 16, 2018 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. SUBCELLULAR LOCALIZATION OF OAT LEAF PROTEASES Table I. Protease Activities at pH 4.5 and 7.5 in Subcellular Fractions (percent of total) and Total Activity in Extracts (,mol glycine equiv * gI * h1) from 4.5 cm Apices of the First Leaffrom 9-d-old Oat Plants Level of detection: 0.3 Amol glycine * g' h'. Extraction Medium Containing Pellet Obtained by No Addition 0.5 M NaCl 0.5 M sucrose Centrifugation at 4.5 7.5 4.5 7.5 4.5 7.5